Evaluation of six methods for external attachment of electronic tags to fish: assessment of tag retention, growth and fish welfare

Abstract External attachment of electronic tags has been increasingly used in fish studies. Many researchers have used ad hoc attachment methods and provided little or no validation for the assumption that tagging itself does not bias animal behaviour or survival. The authors compared six previously published methods for externally attaching acoustic transmitters to fish in a tank holding experiment with black sea bass Centropristis striata (L.). They tracked tag retention, fish growth and external trauma (as a measure of fish welfare) for 60 days. For each of these metrics, the results showed a wide range of responses among tagging treatments. A simple attachment method using a spaghetti tag passed through the dorsal musculature of the fish and tied to the end cap of the transmitter emerged as the preferred option based on high retention, no impact on growth and relatively low detriment to fish welfare. Future field studies using external electronic tagging should consider tag‐related effects that could compromise results when selecting a method for tag attachment.


| INTRODUCTION
Many fish studies using electronic tags have implanted transmitters gastrically or intracoelomically via surgery (Jepsen et al., 2015). Nonetheless, some authors have opted for external attachment out of either necessity or preference based on study design and logistics (Burnett et al., 2020;Burnett et al., 2021). For instance, the antenna of a satellite tag must be free to break the surface of the water in order to transmit. Similarly, satellite archival tags are generally designed to detach after a certain period so they (and the data inside them) can be recovered. Recent technological improvements have increased the options available for telemetry studies, including sensors that record information on light (Block et al., 2005;Seitz et al., 2019), pH (Halfyard et al., 2017;Weinz et al., 2020), depth (Halttunen et al., 2009;Villegas-Ríos et al., 2020), acceleration (Curtis et al., 2015), ambient temperature (Gorsky et al., 2012) and body temperature (Domeier et al., 2019). Some of these sensors (e.g., light) require exposure to the environment and thus must be attached externally to function. It has also been shown that external attachment of acoustic transmitters substantially increases detection range (Dance et al., 2016). Moreover, external attachment is often much faster than surgery (Jepsen et al., 2015) and can be conducted without anaesthesia thereby saving time and money. Finally, some studies must use external tag attachment because surgery would potentially be impractical or bias the results. For example, studies aiming to track fish after they experience barotrauma cannot use surgical implantation because surgery itself would release abdominal gases and perhaps influence post-release behaviour or survival . For these reasons, studies using external attachment of electronic tags have become more common in recent years.
Despite the increase in studies using external attachment, there has been no consensus in the fish telemetry literature on how best to attach transmitters. Many studies have developed methodologies with little or no formal evaluation, though some have performed holding studies usually testing tag retention. Telemetry studies typically assume that tagging does not substantially influence behaviour or survival (Jepsen et al., 2015;Mellas & Haynes, 1985), so ideal methods for tag affixation should maximize retention over the time-scale of the study while minimizing adverse physiological effects to the fish. Methods for transmitter attachment have varied widely; several methods are described below and shown in Figure 1.
1. Single dart. Domeier et al. (2005) used an 8-prong umbrella-shaped plastic dart head that can be tethered to a transmitter with metal wire or cable. The dart is typically inserted into the dorsal musculature of the fish, often between the pterygiophores, and has been used in several studies since (e.g., Bohaboy et al., 2020;Dahl & Patterson, 2020;Dewar et al., 2008).
2. Double dart. Runde and Buckel (2018), Runde et al. (2020) and Wegner et al. (2021) affixed transmitters with two small nylon dart tips attached to a length of galvanized steel wire; the transmitter was fixed to the wire with heat shrink and the dart tips were inserted into the dorsal musculature. In these two studies, it was necessary for transmitters to be affixed at both ends because they contained accelerometers and therefore any dangling or swinging of the tag would bias the readings of this sensor.
3. Cinch-up. Eberts et al. (2018b) and Eberts et al. (2018a) used cinchup tags and created a loop passing once through the muscle beneath the dorsal fin. In both studies, the authors incorporated a loop of dissolvable suture material to hold the transmitter to the cinch-up tag via a hole in the transmitter's end cap to promote intentional short-term loss of the transmitters to minimize the risk of long-term adverse welfare effects. 4. Spaghetti. Capizzano et al. (2016) and Capizzano et al. (2019) used a method involving the use of a spaghetti-style tag passed through the dorsal muscle. They threaded the spaghetti tag through the transmitter end cap and tied it with a single overhand knot. Sweezey et al. (2020) followed the same spaghetti tag method, but added adhesive to the knot in the spaghetti tag to promote retention.

5.
Wire. Bacheler et al. (2021) wrapped stainless steel wire around the transmitter and used marine-grade adhesive and heat shrink tubing to hold the wire to the tag. In this method, the exposed end of the wire was sharpened, passed through the dorsal musculature of the fish and held on the opposite side with a large-diameter aluminium washer and brass crimp. At tagging, the washer and crimp were held firmly against the skin of the fish so the tag was tight against the body.
6. Threaded rod. Bohaboy et al. (2020) created a method where a 2 mm diameter threaded stainless steel rod was passed through the fish and the transmitter end cap; rubber washers cushioned the fish on either side, and lock nuts held the apparatus together.
These studies represent only some of the variety of attachment methods available to researchers conducting telemetry studies, but to the authors' knowledge, no direct comparisons of more than two of these methods exist. Indeed, while some of these studies report F I G U R E 1 Six methods for attaching acoustic transmitters to fish rates of tag loss (e.g., Bohaboy et al., 2020), none to the authors' knowledge empirically examined the impact of tagging on growth and welfare. An experimental comparison of several tag attachment methods would be valuable for designing telemetry studies. Here the authors directly compare the six external attachment methods described above. They provide quantitative results in the form of tag retention and fish growth as well as qualitative and semi-quantitative results regarding animal welfare. To their knowledge, this study is the first to empirically compare multiple tag attachment methods in this way.

| Fish capture, tagging and monitoring
The authors conducted the holding study with black sea bass Centropristis striata (L.). C. striata is an abundant demersal reef fish distributed in the western Atlantic Ocean from Canada to Florida and the Gulf of Mexico, and is important in recreational and commercial fisheries (Musick & Mercer, 1977). Like many species in Serranidae, C. striata is a generalist predator and scavenger and associates with Threaded rod Threaded stainless steel bar (2 mm diameter) sharpened and passed through fish c. 2 cm below anterior insertion of dorsal fin. One end of steel bar passed through silicon cushion (to reduce abrasion) and transmitter end cap; the other end passed through polyethylene and silicon discs. Both ends secured with 6.35 mm nylon-lined stainless steel locknut. (Bohaboy et al., 2020) Note: See Figure 1 for photos of tag attachment methods. recorded for each tag application starting when the fish was removed from the holding tank and stopping when the fish was introduced into its destination tank. Treatments were rotated (six treatments plus an untagged control fish), so that the first seven fish went into tank 1, the next seven went into tank 2, etc. After six tanks each contained one fish of each treatment plus a control fish (7 fish per tank), the authors tagged an additional 12 fish (2 per treatment) and distributed them into the six tanks in rotation to maintain the same overall stocking density (9 fish per tank). Finally, on 3 March 2021, an additional 6 C. striata were tagged (1 per treatment) and introduced into the experimental tanks for a final stocking density of 10 fish per tank.
Thus, each tank contained two fish for each of three treatments, one fish for each of the remaining three treatments and one control fish.
The stocking density of 10 fish per tank was maintained for the duration of the study. All tagging and husbandry was performed under the approval of North Carolina State University IACUC #19-608. The six methods tested in this study were chosen because they were either previously used by an author of the present document or were under the consideration of authors of this study for application in upcoming field studies.
The authors monitored C. striata daily for tag loss and fish mortality. When tag loss was discovered, they removed the affected fish and replaced them on the same day with a freshly tagged fish of the same treatment to increase sample sizes and maintain stocking density.
They tagged a maximum of two replacement fish per treatment, after which fish that had lost their tag were left in the tank and remained untagged. Every 10 days, they weighed and measured each tagged individual plus the never-tagged control fish to monitor growth; previously tagged fish were not weighed and measured. During these sessions, they noted any tag-related lesions or pathologies including scale loss, degree of abrasion and bleeding. External trauma was categorized into four levels: none, mild (abrasion but no scale loss), moderate (scale loss of 1 cm diameter or less; inflamed tissue) or severe (scale loss of more than 1 cm diameter and/or exposed muscle tissue). Starting on day 20 after the first tagging event, they observed signs of infection in some individuals (i.e., abscess at the tag insertion site). They swabbed tag-associated skin lesions from four affected individuals using a commercial bacterial culture col- veterinary-laboratory-investigation-and-response-network) "otheraquatic pathogens" protocol.
The authors concluded the study on 20 April 2021, 60 days after tagging the majority of fish. Tags were removed from most of the stilltagged fish; these individuals were allowed to heal for a 1 week period prior to release in Bogue Sound. A sub-set of still-tagged fish (n = 3 per treatment; total n = 18) were euthanized with an overdose of tricaine methanesulfonate followed by penetrating captive bolt and pithing. Blood was collected immediately postmortem from the caudal haemal arch with heparinized needle and syringe, loaded in haematocrit capillary tubes, and centrifuged in a haematocrit centrifuge to measure packed cell volume (PCV, i.e., erythrocyte volume) and buffy coat (BC) volume (i.e., leukocyte volume) as a rough surrogate for white blood cell count (Kerr, 2010

| Data analysis
The authors assessed how tag retention varied among treatments with Kaplan-Meier survivorship models. Because of the staggered entry of fish into the study, most individuals were observed for 60 days but some more recently tagged fish were observed for as little as 20 days. For the Kaplan-Meier analysis, fish with retained tags were right-censored on the day equal to the duration they were in the study. Retention estimates were compared among treatments and evaluated for whether their 95% C.I. overlapped. Kaplan-Meier analyses were performed and visualized in R (R Core Team, 2021) using the packages "survival" (Therneau, 2015) and "survminer" (Kassambara & Kosinski, 2018).
The authors fitted Bayesian mixed regression models to weight data to examine the effect of tag treatment on growth; these models are analogous to repeated measures analysis of variance.
They elected to use these models given their flexibility, which was necessary due to the complex study design. Although more traditional models may have resulted in similar findings, they preferred the ability to set priors and handle staggered entries and mixed effects in a Bayesian framework. Tag weights were subtracted from total weights obtained during each 10 day measuring period. For this procedure, the response variable was fish wet weight (g) and candidate models included a range of possible predictor variables in addition to treatment (Trt); possible variables were Day (fixed effect), Day*Trt two-way interaction, Tank (random effect) and individual (ID; random effect).
F I G U R E 2 Time (seconds) to apply transmitters to fish in this study using six different attachment methods in Centropristis striata. See Table 1  Models were specified as: Where β terms are variable-specific coefficients, σ is the overall standard deviation in weights, α j is the intercept for tank j, γ j is the intercept for individual j, σ Tank is the tank-specific standard deviation and σ ID is the individual-specific standard deviation. The authors chose lightly informative priors for some terms to apply biologically reasonable constraints and based on prior predictive simulations. They compared fits among models by using leave-one-out cross validation information criterion (LOOIC; Vehtari et al., 2017). For the best model (lowest LOOIC), importance of partial regression coefficients was evaluated by examining 95% credible intervals and checking whether they contained zero. Models were fit using the R package "brms" (Bürkner, 2017).
For examining fish welfare, they coded the four levels of external trauma (none, mild, moderate, severe) as integers 0-3, respectively.
They examined mean and standard error in level of trauma for each tag treatment at each weigh-in period throughout the study. The PCV, BC, TS and histopathology grades were compared among treatment groups.

| Ethical statement
The care and use of experimental animals complied with United States of America animal welfare laws, guidelines and policies as approved by North Carolina State University IACUC #19-608.

| RESULTS
The first two treatments, single dart and double dart, were applied in the lowest amount of time on average, taking 39 and 35 s respectively   F I G U R E 4 Mean and standard error (shaded regions) of external trauma among tagging treatments in Centropristis striata. Trauma was recorded every 10 days throughout the study. The four y-axis categories were coded as 0 (absent), 1 (mild), 2 (moderate) or 3 (severe) for this analysis. See Table 1  No qualitative differences among groups were evident for PCV (combined median 24, range 18%-27%) and plasma TS (combined median 5.6, range 4.4-7.2 g/dl), but BC volumes appeared least for the cinch-up and spaghetti loop (medians 0, range 0%-1%) and greatest for the single dart (median 4, range 0%-4%) and threaded rod (median 3, range 0%-4%).
Weights of C. striata did not differ by tagging method on the day of tagging (Supporting Information Figure S1 in Appendix S1; oneway ANOVA F = 0.642, P = 0.70). The best model for predicting T A B L E 3 Bayesian regression models with untagged fish wet weight as the response variable. An asterisk (*) implies inclusion of both single effects and the two-way interaction between day and treatment. LOOIC is leave-one-out cross validation information criterion. Tank and individual (ID) were included as random effects   (Table 3). In this model, Day was predicted to have a positive effect on weight. The 95% credible intervals for the interaction between Day and treatment were less than zero for the single dart, double dart, cinch-up, wire and threaded rod, indicating that the growth rates of fish tagged with these treatments were lower than that of untagged control fish (Supporting Information Table S1 in Appendix S1). Of the six interaction terms representing growth rate, only the term for the spaghetti loop had a 95% credible interval that overlapped with zero, indicating growth of fish tagged with this treatment was not different from growth of untagged controls. Growth rates for fish tagged with the single dart, double dart and threaded rod appeared appreciably lower than for untagged control fish ( Figure 7).

| DISCUSSION
Understanding the impact of tagging on wild animals is crucial to ensuring the reliability of studies of fish behaviour, movement and survival (Bridger & Booth, 2003;Jepsen et al., 2015). Here the authors demonstrate that several previously used methods for attaching acoustic transmitters to fish can result in adverse effects to growth and welfare, which may impact behaviour or survival thus compromising study results. Tag retention and harmful effects of tagging differed among treatments, and some methods clearly emerged as superior to others.
There was a high degree of variability in tag retention among The full retention of the threaded rods was unsurprising due to their method of affixation using stainless steel lock nuts.
The single dart caused moderate-to-severe trauma to fish beginning on day 10, likely as a result of the relatively large size of the dart tip and length of the external portion of the apparatus. The swinging motion of the tag caused substantial abrasion to the lateral aspect of the fish. External trauma from the double dart was fairly low (mildto-moderate) due to the two attachment points (and therefore no tag movement). The cinch-up and spaghetti tags also caused mild-tomoderate trauma, though tags in these treatments were allowed to swing freely. It appears that the ability of the transmitter to slide along the flexible tag (cinch-up or spaghetti, respectively) resulted in the difference in observed trauma from these treatments as compared to the single dart in which the tag was also free to swing but not able to slide. The spaghetti loop was the only treatment for which fish growth was decidedly not different from growth of the control group, indicating its minimal impact on fish welfare relative to the other treatments. The wire treatment caused moderate-to-severe trauma, largely as a result of the wire and retaining washer. The wire in this treatment was narrow and rigid enough to cut though tissue as the fish moved resulting in eventual loosening of the tag against the body of the fish. Although the credible interval for growth rates of fish tagged with the wire treatment did contain zero, the upper limit was 0.01 indicating that increased sample sizes or a study of longer duration may have resulted in an interval that did not contain zero. The trauma caused by the threaded rod was the most severe of any method examined in this study. The level of abrasion caused by the tag and associated hardware on the left (tag) side of the fish was uniformly severe from day 50 onward. Although other treatments (e. g., single dart) also resulted in severe trauma, the depth of the trauma caused by the threaded rod was greater and appeared to span the entire width of the fish. In addition, the washers on the right side of the fish abraded the scales and skin in most individuals. Indeed, for several fish tagged with the threaded rod, the washers became embedded sub-dermally in muscle tissue. Across treatments, some individuals were able to heal (at least partially) at intervals throughout the study, although this never occurred for fish tagged with the threaded rod.
Though no fish died in this study, they were held in an enclosed system and were therefore not exposed to predators. Oceanic predators may preferentially feed on impaired fish (Bleckmann & Hofmann, 1999), including those that are recovering or traumatized post-tagging (Runde et al., 2020). Therefore, the result of no taginduced mortality may not reflect what would occur in the wild, and the authors suggest that the risk of tag-related predation probably increases with increasing tag-related trauma. Nonetheless, they suspect this bias to be minimal, as some studies have shown no increase in predation on tagged fish (Jepsen et al., 2008).
Long intervals of air exposure can result in higher post-release mortality for fish (Burns et al., 2002;Graves et al., 2016); the speed with which external transmitters can be attached is therefore an important consideration for study design. In this study, there were differences in application time (Figure 2) although almost every individual was tagged in less than 2 min regardless of treatment. The authors echo previous suggestions that researchers practice their tagging method on the study species prior to field deployments to refine mechanics and reduce tagging time as much as possible.
Values for PCV and TS were slightly lower than published values for a sympatric demersal reef fish, invasive red lionfish Pterois volitans L. (PCV median 34, range 27-44; TS median 4.6, range 2.5-7.5 g/dl) (Anderson et al., 2010), but values are comparable for C. striata in the same area sampled immediately following capture (CAH, unpubl. data), indicating that health was not markedly affected by the tagging systems as assessed by these nonspecific indicators. For most applications of haematology in fish studies, BC is usually negligible (0%-1%) and not reported. Larger values for BC thickness, as observed for fish tagged with the single dart (0%-4%) and threaded rod (0%-4%) in the current study, suggest an elevated white blood cell count (Kerr, 2010), consistent with infection and inflammation as also indicated by the higher external trauma scores of these groups.
Long-term tag retention should not be an overriding goal of telemetry studies at the expense of fish welfare (Cooke et al., 2013;Rub et al., 2014). Given the low growth associated with the threaded rod, paired with the associated severe trauma, the authors cannot recommend further use of this method without substantial modification.
Nonetheless, these findings pertain to black sea bass in the laboratory and further testing in the field with other species could find different results. Of the six methods used in this study, the authors recommend the spaghetti loop for use in field studies; the cinch-up could be considered a good second choice. The high tag retention (when properly applied) combined with low external trauma and negligible effects on growth all inspire confidence that the spaghetti loop is minimally invasive and therefore ideal for use in the field. Depending on study goals, dissolvable suture loops should be incorporated into the tagging apparatus to reduce the possibility of indefinite retention of the transmitter and associated adverse physiological effects.
Some transmitters must be attached at two points in order for their sensors to function properly Runde et al., 2020), precluding the use of the spaghetti loop. The only method the authors examined that attached at two locations was the double dart, which did not perform well. Indeed, in both prior in situ studies using this method (Runde et al., 2020;Runde & Buckel, 2018) they observed several instances of tag loss. They recommend that researchers seeking a method that attaches in two places use a modification of their spaghetti loop (e.g., passing the spaghetti tag through the body in two places and affixing to the transmitter with heat shrink) or a method such as was used by Johnson et al. (2015) whereby a cinch-up tag was passed twice through the body and attached to the transmitter via adhesive and plastic cable ties. Future work could compare these and other methods that attach in two places (e.g., Bridger & Booth, 2003;Jacobs et al., 2020).
A common general guideline is that electronic tags should not exceed 2% of the fish's body weight in air (Winter, 1983). Nonetheless, there is substantial evidence that tag weight:body weight ratios of over 10% can be acceptable in telemetry studies (Brown et al., 1999;Jepsen et al., 2005). In this study, ratios of tag weight (including hardware) to body weight on the day of tagging ranged from 1.7% to 6.2% (Supporting Information Figure S2 in Appendix S1). It is conceivable that the fish with a higher ratio were impaired as a result. Field studies seeking to minimize the effects of tag weight could employ smaller tags than those used here though at the potential cost of lower detection range and battery life.
The results with C. striata are considered mostly transferable to other demersal species, and show that some attachment methods (e.g., spaghetti) outperform others (e.g., threaded rod) in virtually every way examined. It remains unclear how these (or other) attachment methods would perform on pelagic fishes, though some results (e.g., among-treatment effects on welfare) are likely to remain constant across species. Where possible, the authors recommend that researchers perform holding studies to evaluate attachment methods prior to field deployment to ensure suitability. In lieu of that, the results could be used as general guidance for field studies, including for pelagic species where holding studies may prove impractical.